CN117737018B - Olefine aldehyde reductase mutant, rhamnosidase mutant, three-enzyme expression strain and application thereof - Google Patents
Olefine aldehyde reductase mutant, rhamnosidase mutant, three-enzyme expression strain and application thereof Download PDFInfo
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- CN117737018B CN117737018B CN202410172865.XA CN202410172865A CN117737018B CN 117737018 B CN117737018 B CN 117737018B CN 202410172865 A CN202410172865 A CN 202410172865A CN 117737018 B CN117737018 B CN 117737018B
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses an enal reductase mutant, a rhamnosidase mutant, a three-enzyme expression strain and application thereof, wherein the enal reductase mutant and the rhamnosidase mutant can overcome the technical problem of insufficient activity of natural enal reductase, and the reaction conditions are mild and can be used for preparing hesperidin dihydrochalcone composition from hesperidin. The conversion rate for preparing hesperidin dihydrochalcone compositions is higher when the enal reductase mutant and the chalcone isomerase mutant are used in combination. The three-enzyme expression strain can simultaneously express a chalcone isomerase CmCHI M mutant, an enal reductase OR mutant and a rhamnosidase RhaB2 mutant, and the chalcone dihydrochalcone glucoside composition is prepared by catalytically converting hesperidin through a double-plasmid expression system of a chalcone isomerase CmCHI M mutant, a recombinant plasmid of the enal reductase mutant and a plasmid of the rhamnosidase mutant.
Description
Technical Field
The invention relates to the technical field of enzyme engineering, in particular to an enal reductase mutant, a rhamnosidase mutant, a three-enzyme expression strain and application thereof.
Background
Neohesperidin dihydrochalcone (NHDC) has a sweetness of 1800 times that of sucrose, but has a bad afterbitter taste similar to licorice, and has a solubility in water of only 0.5 g/L, which limits its application to some extent. This results in the need for the use of neohesperidin dihydrochalcone in the food, pharmaceutical daily use, feed and other industries to be formulated with other sweeteners or taste modifiers. While hesperetin dihydrochalcone glucoside (HCG) is a low calorie non-nutritive sweetener, although its sweetness is about 300-500 times that of sucrose, it is pure in sweetness, free from bitter and aftertaste, and has fruit flavor.
The current preparation method of dihydrochalcone mainly uses a chemical method, uses flavanone as a substrate, and carries out hydrogenation under the action of alkaline conditions and metal catalysts such as platinum, palladium and the like to finally generate the dihydrochalcone. The chemical method is easy to pollute the environment, contains byproducts, has potential safety hazard when being used for preparing food additives, has mild reaction conditions, is environment-friendly and safe, has extremely high specificity, is an ideal preparation way, but has no report on the biosynthesis of dihydrochalcone series products such as hesperidin dihydrochalcone glucoside, hesperetin dihydrochalcone and the like by a one-step method at present.
Alpha-L-rhamnosidase (alpha-L-rhamnosidase, EC 3.2.1.40) is a glycoside hydrolase with wide application prospect, widely exists in plants, animals and microorganisms, can break a glycosidic bond formed by reacting an alcohol hydroxyl group with a hemiacetal in an exo-or endo-mode, and plays an important role in the synthesis and hydrolysis process of a glycoconjugate and a sugar in an organism. The enal reductase (2-alkenal reductase) can catalyze the hydrogenation reduction of unsaturated double bonds in the enal group to generate saturated aldehyde, and then other aldehyde reductases act to generate carbon dioxide and water, so that the reaction of the high-activity unsaturated double bonds with intracellular micromolecular substances is greatly reduced. The natural alpha-L-rhamnosidase and enal reductase have insufficient activity, and the reaction environment is difficult to meet the optimal conditions of the natural enzymes.
Disclosure of Invention
In order to overcome the defects in the prior art, one of the purposes of the invention is to provide an enal reductase mutant which can solve the problem of poor catalytic conversion effect of the existing enal reductase.
The second object of the invention is to provide an application of enal reductase, which is combined with chalcone isomerase mutant, and uses hesperidin as raw material to prepare hesperidin dihydrochalcone composition, and has high conversion rate.
It is a further object of the present invention to provide a strain expressing three enzymes, which can express chalcone isomerase, enal reductase and rhamnosidase simultaneously.
The fourth object of the invention is to provide an application of a three-enzyme expression strain, which can be used for producing hesperidin monoglucoside dihydrochalcone composition.
One of the purposes of the invention is realized by adopting the following technical scheme:
An enal reductase OR mutant, wherein the enal reductase mutant has an amino acid sequence shown in SEQ ID NO:1, the glutamine at position 662 is mutated to cysteine and the aspartic acid at position 462 is mutated to glutamic acid. The mutant was designated OR-Q662A-D462E.
The second purpose of the invention is realized by adopting the following technical scheme:
The enal reductase mutant is used for reducing hesperidin to prepare hesperidin dihydrochalcone.
Preferably, the enal reductase mutant is combined with a chalcone isomerase mutant to catalyze the conversion of hesperidin to prepare the hesperidin dihydrochalcone composition.
The third purpose of the invention is realized by adopting the following technical scheme:
A three enzyme expression strain which is a plasmid expression strain of an enal reductase OR mutant, a rhamnosidase RhaB2 mutant, a chalcone isomerase CmCHI M mutant; wherein, the amino acid sequence of the chalcone isomerase CmCHI M mutant is shown in SEQ ID NO:9, tryptophan at position 120 of chalcone isomerase is mutated into histidine, leucine at position 183 is mutated into tyrosine, and the nucleotide sequence is shown in SEQ ID NO:10 is shown in the figure; the amino acid sequence of the rhamnosidase mutant is shown as SEQ ID NO:5 to isoleucine at position 227 of the rhamnosidase enzyme. So the mutant is mutant RhaB2-W227I. The rhamnosidase mutant is used for preparing hesperetin dihydrochalcone glucoside.
Namely, the three-enzyme expression strain can simultaneously express a chalcone isomerase mutant CmCHI M, enal reductase OR and rhamnosidase RhaB2, wherein OR is OR wild type OR mutant thereof, and RhaB2 is RhaB2 wild type OR mutant thereof.
The fourth purpose of the invention is realized by the following technical scheme:
The use of a trisaccharide expression strain in the preparation of hesperidin dihydrochalcone compositions.
Preferably, the hesperidin dihydrochalcone composition is hesperidin dihydrochalcone glucoside, and the preparation method comprises the following steps: preparing the three-enzyme expression strain into whole-cell enzyme liquid, then adding glucosidase, hesperidin and buffer solution, and reacting at 25-35 ℃ to obtain the hesperetin dihydrochalcone glucoside. The three-enzyme expression strain can be used for preparing the hesperetin dihydrochalcone glucoside under mild conditions, so that the technical problem that the natural enzyme reaction environment is difficult to meet is solved.
The invention actually utilizes the capability of the strain to simultaneously convert the chalcone isomerase CmCHI M, the enal reductase OR and the rhamnosidase RhaB2, and can directly convert the hesperidin into the hesperidin dihydrochalcone glucoside composition by simultaneously expressing the three enzymes. Specifically, it is the enal reductase and chalcone isomerase therein that converts hesperidin to a hesperidin dihydrochalcone composition, and then the rhamnosidase enzyme converts hesperidin dihydrochalcone to a hesperetin dihydrochalcone glucoside composition.
Specifically, the construction method of the three-enzyme expression strain comprises the following steps:
(1) Constructing recombinant plasmids of chalcone isomerase CmCHI M, enal reductase OR and a vector to obtain a chalcone isomerase-enal reductase recombinant plasmid; wherein, the vector is escherichia coli, namely pET28a-CmCHI M -OR plasmid;
(2) Constructing a rhamnosidase plasmid; wherein the vector is escherichia coli and is marked as pET32a-RhaB2 plasmid;
(3) Constructing and screening a composition of a chalcone isomerase mutant CmCHI M and an enal reductase mutant OR-Q662A-D462E in the pET28a-CmCHI M -OR plasmid obtained in the step (1); designated as CmCHI M -OR-Q662A-D462E;
(4) Constructing and screening a rhamnosidase mutant RhaB2-W227I in the rhamnosidase plasmid obtained in the step (2);
(5) And co-converting the composition of the chalcone isomerase mutant and the enal reductase mutant and the rhamnosidase mutant into a carrier, and screening positive transformants in a culture medium to obtain the three-enzyme expression strain.
It should be noted that the carrier combinations of the chalcone isomerase CmCHI M, the enal reductase OR, and the rhamnosidase RhaB2 include, but are not limited to, the above methods, OR the three enzymes may be combined by using different carriers, OR the combination of the three enzymes may be performed after the combination of the two enzymes. The combination mode of the three enzymes does not influence the expression effect of the three enzymes.
Preferably, in the step (1), the construction method of the recombinant chalcone isomerase-enal reductase plasmid comprises the following steps: the method comprises the steps of taking a synthetic chalcone isomerase gene with an amino acid sequence shown as SEQ ID NO. 1 and a nucleotide sequence shown as SEQ ID NO. 2 as a template, synthesizing two primers shown as SEQ ID NO. 3 and SEQ ID NO. 4 in a sequence table, respectively adding two digestion sites of Nde I and EcoR I at two ends of the synthesized primers, carrying out PCR, separating a PCR product by agarose gel electrophoresis, connecting the recovered PCR product into plasmids of chalcone isomerase and a vector, screening positive transformants, and obtaining the recombinant plasmid of the chalcone isomerase-enal reductase.
Preferably, in the step (2), the method for constructing the rhamnosidase plasmid comprises the following steps: the method comprises the steps of taking a synthetic gene rhamnosidase RhaB2 with an amino acid sequence shown as SEQ ID NO. 5 and a nucleotide sequence shown as SEQ ID NO. 6 as a template, synthesizing two primers shown as SEQ ID NO. 7 and SEQ ID NO. 8 in a sequence table, respectively adding EcoR I and Not I enzyme cutting sites at two ends of the synthesized primers, carrying out PCR, separating PCR products by agarose gel electrophoresis, connecting the recovered PCR products into a pET32a vector, screening out positive transformants, and obtaining recombinant plasmids named pET32a-RhaB2 plasmids.
Compared with the prior art, the invention has the beneficial effects that:
(1) The enal reductase mutant can overcome the technical problem of insufficient activity of natural enal reductase, has mild reaction conditions, and can be used for preparing hesperidin dihydrochalcone composition from hesperidin. The conversion rate for preparing hesperidin dihydrochalcone compositions is higher when the enal reductase mutant and the chalcone isomerase mutant are used in combination.
(2) The rhamnosidase mutant can overcome the technical problem of insufficient activity of natural rhamnosidase, has mild reaction conditions, can be used for converting hesperidin dihydrochalcone into a hesperetin dihydrochalcone glucoside composition, and has high conversion rate.
(3) The three-enzyme expression strain can simultaneously express a chalcone isomerase CmCHI M mutant, an enal reductase OR mutant and a rhamnosidase RhaB2 mutant, and the hesperidin dihydrochalcone glucoside composition is prepared by catalyzing and converting hesperidin through a double-plasmid expression system of recombinant plasmids of the chalcone isomerase CmCHI M mutant, the enal reductase OR mutant and plasmids of the rhamnosidase RhaB2 mutant.
(4) The application of the three-enzyme expression strain can directly convert hesperidin into a hesperetin dihydrochalcone glucoside composition. Specifically, it is the enal reductase and chalcone isomerase therein that converts hesperidin to a hesperidin dihydrochalcone composition, and then the rhamnosidase enzyme converts hesperidin dihydrochalcone to a hesperetin dihydrochalcone glucoside composition. Fills the blank that hesperidin is not taken as a raw material at the present stage, and hesperidin is directly catalyzed and converted into hesperidin dihydrochalcone, hesperetin dihydrochalcone glucoside and hesperetin dihydrochalcone by a biological method.
Drawings
FIG. 1 is a diagram of a three-dimensional structural model of a chalcone isomerase-enal reductase combination mutant;
FIG. 2 is a diagram of a three-dimensional structural model of a rhamnosidase mutant;
FIG. 3 is a schematic diagram showing the enzyme activities of a chalcone isomerase-enal reductase combination mutant and a wild-type enal reductase;
FIG. 4 is a chromatographic analysis of the rhamnosidase mutant and wild-type rhamnosidase reaction product;
FIG. 5 is a chromatographic analysis of the catalytic preparation of hesperetin dihydrochalcone glucoside by a three enzyme expression strain;
FIG. 6 is a diagram of a chromatographic analysis of the catalytic preparation of hesperetin dihydrochalcone by a three enzyme expression strain.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the embodiments described below or technical features may be arbitrarily combined to form new embodiments. The following are specific examples of the present invention, in which raw materials, equipment, etc. used are available in a purchase manner except for specific limitations.
In the following examples, the specific conditions are specified by the conventional experimental conditions or the experimental conditions suggested by the manufacturer, and the various reagents involved in the examples are commercially available unless otherwise specified. The experimental method of molecular biology, which is not specifically described in this example, can be referred to the "guidelines for molecular cloning experiments".
Example 1
Relates to the construction of related plasmids of the patent:
construction of recombinant plasmids pET28a-CmCHI M -OR of E.coli by chalcone isomerase and enal reductase
The nucleotide sequence of the primer is shown as SEQ ID NO. 1, the olefine aldehyde reductase OR mutant synthetic gene is shown as SEQ ID NO. 2, two primers shown as SEQ ID NO. 3 and SEQ ID NO. 4 in a sequence table are synthesized by using the olefine aldehyde reductase OR mutant synthetic gene as a template, and Nde I and EcoR I enzyme cutting sites are respectively added at two ends of the synthesized primers to carry out PCR. The DNA polymerase is KOD DNA polymerase with high fidelity of Bao bioengineering Co., ltd. The PCR amplification procedure was: 94 ℃ for 2min; 15s at 94 ℃, 30s at 58 ℃ and 2min at 72 ℃ for 35 cycles; the temperature was reduced to 10℃for 10min at 72 ℃. The DNA was recovered from the agarose gel using Axygen Gel Extraction Kit (AEYGEN). The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into Nde I and EcoRI double digested pET28a-CmCHI M vector using T4 DNA ligase from ThermoFisher. Positive transformants were selected on Kan-resistant medium and the recombinant plasmid obtained was designated pET28a-CmCHI M -OR. Wherein, the amino acid sequence of the chalcone isomerase mutant is shown in SEQ ID NO:9, the nucleotide sequence of which is shown as SEQ ID NO: shown at 10.
Construction of the (two) rhamnosidase plasmid pET32a-RhaB2
The two primers of SEQ ID NO. 7 and SEQ ID NO. 8 in the sequence table are synthesized by taking a rhamnosidase RhaB2 mutant with an amino acid sequence shown as SEQ ID NO. 5 and a nucleotide sequence shown as SEQ ID NO. 6 as a template, and EcoR I and Not I cleavage sites are respectively added at two ends of the synthesized primers for PCR. The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into EcoR I and Not I double digested pET32a vector using T4 DNA ligase. Positive transformants were selected on medium containing Amp resistance and the recombinant plasmid obtained was designated pET32a-RhaB2.
Example 2
Construction of chalcone isomerase-enal reductase (CmCHI M -OR) mutant and rhamnosidase RhaB2 mutant:
And carrying out artificial intelligence modeling on OR by utilizing AlphaFold2, and evaluating the model to obtain a reliable three-dimensional structure model according to the evaluation of the scoring standard, wherein the three-dimensional structure model is shown in figure 1. Multiple active sites are obtained by simulating a docking ligand, a mutant is constructed, enzyme activity detection is carried out on the mutant, and the mutant CmCHI M -OR-Q662A-D462E is obtained through screening, namely, the mutation of glutamine at 662 to cysteine and the mutation of aspartic acid at 462 to glutamic acid.
And carrying out artificial intelligence modeling on RhaB2 by AlphaFold, evaluating the model, and obtaining a reliable three-dimensional structure model according to the evaluation standard, wherein the three-dimensional structure model is shown in figure 2. Multiple active sites are obtained through simulating a docking ligand, a mutant is constructed, enzyme activity detection is carried out on the mutant, and the mutant RhaB2-W227I is obtained through screening, namely, the 227 th tryptophan is mutated into isoleucine.
Example 3
Comparison of mutant CmCHI M -OR-Q662A-D462E with wild-type enzyme activity:
and (3) respectively culturing the mutant CmCHI M -OR-Q662A-D462E and the wild type in an LB culture medium containing Kan until the OD600 is 0.6-1.0, adding 0.2 mmol/L IPTG to induce the strain at 20 ℃ for 18 h, and then obtaining 2 parts of whole-cell crude enzyme solution with equal concentration through the steps of centrifugation, supernatant removal, water washing, bacterial cell resuspension and the like. Finally, the hesperidin with the final concentration of 2mM is added, the whole cell catalytic reaction is carried out at 30 ℃ for 24 h, and the conversion rate of the hesperidin dihydrochalcone is detected by HPLC, as shown in figure 3, the result shows that the enzyme activity of CmCHI M -OR-Q662A-D462E is improved, the conversion rate is improved from 8.7% to 75.4%, and the conversion rate is improved by more than 8.6 times compared with the wild strain.
Example 4
Comparison of mutant rhaB2-W227I with wild type enzyme activity:
And (3) respectively culturing the mutant RhaB2-W227I and the wild rhamnosidase RhaB2 in an LB culture medium containing Kan until the OD 600 is 0.6-1.0, adding 0.2 mmol/L IPTG to induce the strain at 20 ℃ for 18 h, and then obtaining 2 parts of whole-cell crude enzyme solution with the same concentration through the steps of removing the supernatant by centrifugation, washing the thalli by water, re-suspending the thalli and the like. Finally, the hesperidin dihydrochalcone with the final concentration of 2mM is added, the whole cell catalytic reaction is carried out at 30 ℃ for 24h, and the conversion rate of hesperetin dihydrochalcone glucoside is detected by HPLC, as shown in figure 4, the result shows that the enzyme activity of RhaB2-W227I is improved, the conversion rate is improved from 11.5% to 81.3%, and the conversion rate is improved by more than 7 times compared with the wild rhamnosidase RhaB2 strain.
Example 5
Construction and application of a double plasmid expression strain of a combination mutant plasmid (pET 28a-CmCHI M -OR-Q662A-D462E) of an enal reductase mutant and a chalcone isomerase mutant and a rhamnosidase mutant plasmid (pET 32A-RhaB 2-W227I):
Co-transforming pET28a-CmCHI M -OR-Q662A-D462E and pET32A-RhaB2-W227I into BL21, screening positive transformants by using a culture medium containing Amp and Kan double resistance, and obtaining a double plasmid expression strain named COR, namely a three-enzyme expression strain.
Culturing COR in LB culture medium containing Amp and Kan until OD600 is 0.6-1.0, adding 0.2 mmol/L IPTG to induce 18 h at 20 ℃, and performing steps such as centrifugation to remove supernatant, water washing thalli, re-suspending thalli and the like to obtain the whole cell crude enzyme liquid. Finally, the hesperidin with the final concentration of 2mM is added, the whole-cell catalytic reaction is carried out at 30 ℃ for 24 h, and the conversion rate of hesperidin dihydrochalcone and hesperetin dihydrochalcone glucoside is detected by HPLC, as shown in figure 5, the result shows that the hesperidin can be converted into hesperetin dihydrochalcone glucoside by a COR strain in one step, and the conversion rate can reach 71.5%.
Example 6
Application of COR in combination with commercial glucosidase:
200U/mL commercial glucosidase and 2mM hesperidin are added into whole-cell enzyme solution expressed by COR as raw materials, pH 4.6 citric acid-phosphate is taken as buffer solution, and the mixture is reacted for 24 hours at 30 ℃, as shown in FIG. 6, and the conversion rate of the hesperetin dihydrochalcone glucoside is 69.7 percent through detection.
Comparative example 1
Comparative example 1 differs from example 5 in that: the strain of comparative example 1 only expresses rhamnosidase and chalcone isomerase, i.e. it is only transformed from a plasmid of the combination of rhamnosidase RhaB2 and chalcone isomerase mutant CmCHI M. Culturing the strain in LB culture medium containing Kan until the OD600 is 0.6-1.0, adding 0.2 mmol/L IPTG to induce the strain at 20 ℃ for 18 h, centrifuging to remove supernatant, washing thalli, re-suspending thalli and other steps to obtain whole cell crude enzyme liquid, adding hesperidin with the final concentration of 2mM, and carrying out whole cell catalytic reaction at 30 ℃ for 24h, wherein the conversion rate of the hesperetin dihydrochalcone glucoside is 0%.
Comparative example 2
Comparative example 2 differs from example 5 in that: the strain of comparative example 2 only expresses chalcone isomerase mutant and enal reductase, namely, the strain is only transformed by a plasmid combined by chalcone isomerase mutant CmCHI M and enal reductase OR mutant, when the strain is cultured in LB culture medium containing Kan until OD600 is 0.6-1.0, 0.2 mmol/L IPTG is added to induce 18. 18 h at 20 ℃, then the steps of supernatant removal, bacterial body washing, bacterial body resuspension and the like are carried out through centrifugation to obtain whole cell crude enzyme liquid, finally, hesperidin with the final concentration of 2mM is added, and the whole cell catalytic reaction is carried out at 30 ℃ for 24 h, wherein the conversion rate of the whole cell crude enzyme liquid into hesperetin dihydrochalcone glucoside is 0%.
Comparative example 3
Comparative example 3 differs from example 5 in that: the strain of comparative example 3 only expresses rhamnosidase and enal reductase, i.e. it is only transformed with a plasmid of the combination of rhamnosidase mutant RhaB2 and enal reductase OR mutant. Culturing the strain in LB culture medium containing Kan until OD 600 is 0.6-1.0, adding 0.2 mmol/L IPTG to induce 18 h at 20 ℃, centrifuging to remove supernatant, washing thalli, re-suspending thalli and other steps to obtain whole cell crude enzyme liquid, adding hesperidin with the final concentration of 2mM, and carrying out whole cell catalytic reaction 24h at 30 ℃ to convert the hesperetin dihydrochalcone glucoside into the hesperetin dihydrochalcone glucoside with the conversion rate of 0%.
In summary, as shown in examples 5 and comparative examples 1 to 3, when only one or two of chalcone isomerase, enal reductase and rhamnosidase are expressed in the strain, it is impossible to convert hesperidin to hesperidin dihydrochalcone glucoside. Because chalcone isomerase must be used in combination with enal reductase to convert hesperidin to hesperidin dihydrochalcone, whereas rhamnosidase can only convert hesperidin dihydrochalcone to hesperidin dihydrochalcone hesperetin dihydrochalcone glucoside. Therefore, the three enzymes can be combined to convert the hesperidin into the hesperidin dihydrochalcone glucoside.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (3)
1. An enal reductase mutant, which is characterized in that the enal reductase mutant has an amino acid sequence shown in SEQ ID NO:1, wherein glutamine at position 662 of the enal reductase is mutated to cysteine and aspartic acid at position 462 is mutated to glutamic acid.
2.A trisaccharase expression strain, characterized in that the strain is a plasmid expression strain of the enal reductase mutant, rhamnosidase mutant and chalcone isomerase mutant of claim 1; wherein, the amino acid sequence of the chalcone isomerase mutant is shown in SEQ ID NO:9, the nucleotide sequence of which is shown as SEQ ID NO:10 is shown in the figure; the amino acid sequence of the rhamnosidase mutant is shown as SEQ ID NO:5 to isoleucine at position 227 of the rhamnosidase enzyme.
3. Use of a strain expressing a three enzyme according to claim 2 for the preparation of a hesperidin dihydrochalcone composition, wherein the hesperidin dihydrochalcone composition is hesperetin dihydrochalcone glucoside, a method for the preparation thereof comprising the steps of: preparing the three-enzyme expression strain into whole-cell enzyme liquid, then adding glucosidase, hesperidin and buffer solution, and reacting at 25-35 ℃ to obtain the hesperetin dihydrochalcone glucoside.
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